Process and arrangement for confocal microscopy

ABSTRACT

A process for confocal microscopy is disclosed  in which laser light is coupled into a microscope beam path, directed successively with respect to time onto different locations of a specimen, and an image of the scanned plane is generated from the light reflected and emitted by the irradiated locations. A change in the spectral composition and in the intensity of light is  are carried out during the deflection of the laser beam from location to location, while the deflection continues in an uninterrupted manner. In this way , so that at least two adjacent locations of the specimen located next to one another  are acted upon by light with different spectral characteristics and by laser radiation of different intensity. By periodically interrupting the coupling in of the laser light during the deflection of the microscope beam path, it is made possible that  only selected portions of the image field are acted upon by the laser radiation. A laser scanning microscope for carrying out this process is also disclosed. A laser scanning microscope for carrying out this process is also disclosed.

BACKGROUND OF THE INVENTION

a) Field of the Invention

The invention is directed to a process for confocal microscopy in whichlaser light of different spectral ranges is coupled into a microscopebeam path deflected in at least two coordinates and is directedsuccessively with respect to time onto locations of a specimen, whereinthe specimen is acted upon, location by location and line by line, bythe laser light in at least one plane and an image of the scanned planeis generated from the light reflected and/or emitted by the irradiatedlocations. The invention is further directed to a laser scanningmicroscope for carrying out this process.

b) Description of the Related Art

While conventional light microscopy only enables the optical acquisitionof one imaging plane, confocal microscopy, as a special furtherdevelopment of light microscopy, offers the possibility of imaging andmeasuring microstructures also in the Z spatial axis. With lightmicroscopy, it is not possible, for example, to gain an impression ofthe spatial structure of the rough surface of a specimen at highmagnification because only a small area of the specimen can be shown insharp focus, while details located deep in the surface are imaged in ablurry manner because of the high scattered light component anddeficient axial resolution.

In confocal laser scanning microscopy, on the other hand, the scatteredlight is extensively eliminated and only the structures located in thefocal plane of the objective are imaged. If the radiation is focused ondifferent planes, three-dimensional images of a specimen can becalculated from the scanning of these planes which are staggered in thedirection of the Z-axis.

For this purpose, a first pinhole is imaged in the object plane so as tobe reduced in a punctiform manner using lasers as an illuminationsource. The punctiform laser beam is moved over the specimen in a rasterpattern from location to location and line by line by means ofdeflecting mirrors. The light reflected and/or emitted by the specimenis focused through the microscope objective onto a second pinhole whichis arranged so as to be conjugated with respect to the first pinhole. Asa result of the arrangement of these two pinholes, only information fromthe focal plane reaches one or more detectors which are arrangedfollowing the second pinhole.

The scattered light occurring above and below the focus is eliminated bythe second pinhole. The information determined by two-dimensionaldeflection from a plurality of imaging planes located one above theother is stored and subsequently processed to form images.

This principle of confocal laser scanning microscopy is described, forexample, in Schroth, “Konfokale Laser-Scaning-Mikroskopie, eine neueUntersuchungsmethode in der Materialprüfung [Confocal Laser ScanningMicroscopy, a new method of investigation in materials testing]”,Zeitschrift Materialprüfung, volume 39 (1997), 6, pages 264 ff.

Further, it is known from “Mitteilungen für Wissenschaft und Technik”,volume II, no. 1, pages 9-19, June 1995, to use either individuallasers, each having one wavelength, or “multi-line” mixed gas laserswith a plurality of usable wavelengths as illumination source in laserscanning microscopes. This opens up the possibility of utilizingconfocal microscopy for fluorescence technique in addition to theclassic contrasting processes of bright field, phase contrast andinterference contrast. The basis for this consists in that differentfluorochromes whose excitation and emission wavelengths lie in differentspectral regions allow structures to be shown in a plurality offluorescence colors simultaneously. Accordingly, depending on thespectral characteristics of different dye molecules, conclusions may bereached about physiological parameters in addition to morphologicalinformation. When the confocal microscope is used for fluorometricprocesses, information can be derived concerning changes in theconcentration of ions and molecules. In this connection, other importantindicators are those which show a shifting of the excitation andemission spectrum in addition to the intensity dependence and, in thisregard, enable a quantification of ion concentrations. Also proposed inthis connection is the photobleaching method in which a definednonuniformity is generated in order to be able to obtain informationabout the object such as fluidity and diffusion through the dynamics ofthe equilibrium which is subsequently initiated.

It is known from the above-cited publication to use Ar-Kr lasers forfluorescence excitation in the visible spectral region with lines 488nm, 568 nm and 647 nm. These lines are combined in a laser beam andsupplied to the scanning device via light-conducting fibers. An Ar laserwith wavelengths 351 nm and 364 nm is suggested for excitation in the UVrange. Coupling into the scanning device is also carried out in thisinstance via light-conducting fibers.

The processes and arrangements described herein can be utilized foracquiring 3D data records which allow, for example, a reliablecorrelation of spatial cell structures and tissue structures within amicroarchitecture or the localization of a plurality of gene sites inchromosomes in FISH experiments.

However, a disadvantage consists in that the respective specimen isacted upon over the entire scanning region by the laser radiation thatis generated in the laser module and coupled into the scanning unit. Theentire scanning region is therefore exposed to a relatively highradiation loading which leads to unwanted effects and insufficientresults particularly when investigating living organisms.

A further disadvantage consists in that radiation emitted and/orreflected from a determined location on a specimen cannot be detectedand evaluated in a definite manner when the specimen is excited withdifferent wavelengths such as those of the above-mentioned laser lines,since the “bleed-through” effect occurs between the individual spectrallines.

OBJECT AND SUMMARY OF THE INVENTION

On this basis, the primary object of the invention is to further developa process for laser scanning microscopy of the type described above insuch a way that the radiation loading of the specimen is reduced and amore precise image evaluation is achieved.

According to the invention, this object is met in that a change in thespectral composition and/or in the intensity of light is carried outduring the deflection of the laser beam from location to location. Thisis effected either in that the coupling in of individual spectralcomponents or of a plurality of spectral components or the radiation ofthe light in its entirety is periodically interrupted or in thatindividual spectral components or a plurality of spectral components areperiodically coupled into the microscope beam path additionally, whilethe deflection of the microscope beam path continues in an uninterruptedmanner.

In this way, at least two locations located next to one another on thespecimen are acted upon by light with different spectral characteristicsand/or by laser radiation of different intensity. By periodicallyinterrupting the coupling in of the laser light during the deflection ofthe microscope beam path, it is made possible that only selectedportions of the image field are acted upon by the laser radiation.

The specimen is protected in that only the areas of the specimenrelevant for image evaluation are acted upon by laser radiation of highintensity.

In a preferred construction variant of the process according to theinvention, the spectral composition and/or intensity of the laser lightis changed during the scanning of a plurality of locations which arelocated adjacent to one another and thus form a scanning line. In thisconnection, the deflection can be carried out over the locations of thisline repeatedly in the same direction or also bidirectionally. It isprovided according to the invention, for example, that the change in thespectral composition or in the intensity is always carried out withreference to the same locations lying adjacent to one another in thisline during every scan over the locations in this line, regardless ofwhether this scan is carried out in the same direction or in theopposite direction, so that the quality of the image evaluation isincreased while the energy introduced into the specimen remains limited.In this way, it is achieved at the same time that individual adjacentlocations of the specimen can be observed without bleed-through ofindividual spectral regions into one another.

The different spectral composition of the laser radiation coupled intothe microscope beam path is achieved, for example, in that the radiationprovided by a plurality of line lasers, e.g., with wavelengths of 633nm, 568 nm, 543 nm, 514 nm, 488 nm and 458 nm, is coupled in as requiredor depending on the characteristics of the specimen to be evaluated withan individual wavelength, with a selection of a plurality of individualwavelengths or with all available individual wavelengths. In addition tothis radiation in the VIS range, additional wavelengths in the UV range,for example, 351 nm and 364 nm, can be provided for coupling in.

In preferred constructions of the invention, the laser radiation iscoupled into the microscope beam path via single-mode fibers so as tomaintain polarization. The respective laser lines provided for radiationare advantageously adjusted to a desired brightness with anacousto-optic tunable filter (AOTF) which can also be followed by anacousto-optic modulator (AOM). The respective laser wavelength isadapted to the microscope objective placed in the beam path for both theUV and the VIS region by variable beam collimation.

A further preferred construction of the process according to theinvention consists in that the light reflected and/or emitted by everyindividual irradiated location of the specimen is evaluated with respectto its spectral characteristics and intensity, wherein the evaluation iscarried out synchronously with the irradiation of the same location andwhile taking into consideration the spectral composition and/orintensity of the laser light by which this location is irradiated. Thismakes it possible to evaluate the scanned portion of the specimen withrespect to individual locations, which leads to a very high resolutionand to the highest possible accuracy in the evaluation of the image.

It also lies within the framework of the invention that the laser lightreflected and/or emitted by every individual irradiated location isdetected with a plurality of detection channels, wherein the individualdetection channels are arranged for receiving different spectralcomponents. This provides very good conditions for the examination ofmultifluorescence specimens, and identical optical sections can begenerated via every detection channel with simultaneous reception ofmultifluorescence specimens.

In this connection, it is provided according to the invention that thespectral composition and/or the intensity of the laser light which iscoupled into the microscope beam path corresponds to the excitationradiation of a fluorescence dye contained in the specimen or applied tothe specimen and the individual detection channels are configured forthe reception of the emission radiation proceeding from the fluorescencedye. This makes it possible to generate laser light for the excitationof different fluorescence dye and to draw conclusions from the detectionconcerning the distribution of these fluorescence dyes on or in thespecimen.

Another very preferable construction of the invention consists in thatan evaluation of the spectral composition and/or of the intensity of thecoupled in laser light is carried out in a continuous manner and theevaluation findings for the laser radiation directed to a determinedlocation are mathematically linked with the evaluation findings for thelight reflected and/or emitted by this location. As a result of thislink, for example, the deflection position of the microscope beam pathfor two adjacent locations can be determined according to thecoordinates x, y, z for which differences in the spectralcharacteristics of the light reflected and/or emitted from theselocations which go beyond a predetermined threshold value can bedetected during evaluation, wherein, based on these differences,conclusions can be reached concerning the presence of an opticalboundary layer between these two locations. These deflection positionsare stored, according to the invention, and taken as a basis for thecalculation of surface areas and/or volumes enclosed by optical boundarylayers within the specimen.

Further, with the deflection positions which are obtained and stored inthis way, it is possible to determine and preset adjustment signals forthe spectral composition and/or the intensity of the laser light forirradiation of these locations during a subsequent scan cycle, so thatan automatic optimization is achieved in the image evaluation whiletaking into account the optical characteristics of the specimen and ofthe fluorescence dye.

In particular, the process according to the invention can be used in anadvantageous manner for photobleaching, as it is called. In thisconnection, a selected area of a specimen can be acted upon initially bya relatively high radiation intensity during scanning, therebyinitiating a bleaching process. With the scan cycles followingimmediately thereafter, the reactions taking place are opticallydetected and evaluated, wherein information can be obtained about thedynamic processes such as diffusion and transport processes taking placein the specimen substance immediately after the bleaching process.

For this purpose, the scanning must be carried out with a very high timeresolution, which is achieved, according to the invention, by switchingbetween different intensities and different spectral compositions of thelight impinging on individual locations of the specimen, wherein thisswitching is carried out with sufficient speed in a synchronous mannerwith respect to the deflection of the beam.

The fast switching between different intensities and different spectralcompositions of the laser radiation is carried out with an acousto-optictunable filter (AOF) which, in an analogous but substantially fastermanner, takes over the function of different filters which can besubstituted for one another in the beam path and which, further, canalso modulate the intensity of individual laser lines or optionalcombinations of lines in a highly dynamic manner with respect to time.

The manner of operation and application of the AOTF is thoroughlydescribed, for example, in String, Kenneth, R., “Wavelength Selectionfor Illumination in Fluorescence Microscopy”, NIH, LKEM, Building10/6N309, Bethesda, MD 20892, April 1993. Further, concrete applicationexamples for the AOTF are described in U.S. Pat. Nos. 5,444,528,5,377,003 and 5,216,484.

The synchronization in time between the driving of the AOTF formodulation of the laser radiation and the driving of the scanning devicefor beam deflection is achieved in that determined control signals forthe AOTF are correlated with the control signals supplied to thescanning device by the driving device. Thus, the scanning device andAOTF are always driven synchronously, i.e., the control pulses for theAOTF are, with respect to time, always added to the output of a controlpulse for the scanning device.

On the other hand, this means that a characteristic intensity and/orspectral composition of the light can be assigned to every deflectionposition and accordingly to every location of the specimen.

In this respect, the circuit arrangements for executing the process areoptimized by the AOTF with respect to very short transit times of thecontrol pulses from output to switching of beam modulation. Thesetransit times are in the range of <10 ms. In a variant of the process,when controlling the AOTF or the scanning device, rate action times orlead times are calculated beforehand for switching the intensity andspectral composition and/or for deflection, so that precisely theintended location is also irradiated with the intended radiationintensity and spectral composition.

The invention is further directed to a laser scanning microscope forcarrying out the process steps described above, with a laser module forgenerating laser light with different selectable spectral components,with single-mode fibers for coupling the laser light into the microscopebeam path, with a scanning device which deflects in at least twodimensions, with a microscope objective which focuses the laser light ona specimen, with a plurality of detectors for the reception of differentspectral components of the light reflected and/or emitted by thespecimen, and with an evaluation circuit which is connected subsequentto the outputs of the detectors.

In a laser scanning microscope of this kind, according to the invention,a plurality of individually controllable single-wavelength and/ormultiple-wavelength lasers are provided in the laser module, the lasermodule is followed by a beam combiner, an acousto-optic tunable filter(AOTF) and/or an acousto-optic modulator (AOM), the single-mode fiber isfollowed by collimating optics whose distance from the respective end ofthe fiber can be changed and which are coupled with drivable adjustingdevices. Photomultipliers (PMT) are provided as detectors, each of whichis associated with a reflection band or emission band and accordinglywith a detection channel. Filters and/or color splitters which arearranged on splitter wheels and which can be substituted for one anotherby rotating the splitter wheels are provided for branching the radiationproceeding from the specimen into individual detection channels, whereinevery splitter wheel is likewise coupled with a controllable adjustingdevice. Further, the control inputs of the laser module, AOTF, AOM,scanning device and adjusting devices for the splitter wheels andcollimating optics are connected with the outputs of the evaluationcircuit.

In a construction variant of the laser scanning microscope, themicroscope beam path directed on the specimen is branched and one of thebranches is directed to an optoelectronic receiver whose output islikewise connected with the driving unit.

Further, it is provided in a preferred construction variant that amathematical linking of the output signals of the optoelectronicreceiver with the output signals of the PMT and/or with the deflectionsignals for the scanning device is carried out in the evaluationcircuit, wherein optimized adjusting signals for the laser module, AOTF,AOM, scanning device and for the adjusting device are made available atthe output of the evaluation circuit.

The invention will be explained more fully hereinafter with reference toan embodiment example.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings:

FIG. 1 shows the basic construction of a laser scanning microscope; and

FIG. 2 shows a schematic illustration of the deflection of the laserlight over the individual locations of a specimen.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

FIG. 1 shows a laser module 1 which is outfitted with lasers 2, 3 and 4for generating laser light in the visible range with wavelengths of 633nm, 543 nm and 458 nm. By mean of a plurality of beam combiners 5, anAOTF 6 and a fiber 7, the radiation emitted by these lasers is coupledinto a scanning device 8 which is outfitted with a unit 9 deflectingbeams in the x and y coordinates.

A UV laser whose light is coupled into the scanning device 8 via an AOTF11 and a light-conducting fiber 12 is provided in a second laser module10.

In the two beam paths, collimating optics 13 are provided subsequent tothe light-conducting fibers 7 and 12, wherein the distance between thecollimating optics 13 and the respective end of the fiber can be changedand the collimating optics 13 are coupled for this purpose with acontrollable adjusting device (not shown in the drawing).

The laser radiation is coupled into the beam path of the schematicallyshown microscope 15 by the beam-deflecting device 9 through a scanningobjective 14 and is directed on a specimen 16. For this purpose, thelaser radiation passes through a tube lens 17, a beam splitter 18 andthe microscope objective 19.

The light returned (reflected and/or emitted) by the irradiated locationat the specimen travels back through the microscope objective 19 to thebeam-deflecting device 9, then passes through a beam splitter 20 and,after being branched into a plurality of detection channels 22, isdirected by the imaging optics 21 onto photomultipliers 23, each ofwhich is associated with a detection channel 22. For the purpose ofbranching into the individual detection channels 22, the light isdirected from a deflection prism 24 to dichroitic beam splitters 25.Emission filters 27 and pinholes 26 are provided in every detectionchannel 22, wherein the latter are adjustable in the direction ofradiation and vertical thereto and also in diameter.

The outputs of the photomultipliers 23 lead to the signal inputs of anevaluation circuit 28 which is connected in turn with a driving device29. The outputs of the driving device 29 are connected with the signalinputs of the laser modules 1 and 10 and with signal inputs of theadjusting devices for influencing the position of optical elements andcomponent groups such as, for example, the position of the collimatingoptics 13, pinholes 26 and the like (not shown in detail).

For example, the laser radiation that is coupled into the scanningdevice 8 is branched through a beam splitter 30, one of the branchesbeing directed to an optoelectronic receiver 31, wherein a plurality ofline filters 32 which are arranged on filter wheels and can be exchangedwith one another by rotating the filter wheels and neutral filters 33which can likewise be exchanged with one another are arranged in frontof the optoelectronic receiver 31. The output of the receiver 31 islikewise applied to a signal input of the evaluation circuit 28. Thefilter wheels on which the line filters 32 and the neutral filters 33are arranged are coupled with adjusting devices whose control inputs areconnected with signal outputs of the driving device 29 (not shown in thedrawing).

During operation of the laser scanning microscope, the optical axis 38of the microscope beam path is guided through the scanning device 8, asis illustrated in FIG. 2, in the direction of coordinate X from locationto location and in the direction of coordinate Y from line to line in araster pattern over the object plane 34 to be scanned, wherein thedetail 35 of a specimen which is to be evaluated lies in this objectplane 34.

In the prior art, laser light was previously coupled into the microscopebeam path with a spectral composition and intensity which remained thesame during scanning. As a result, a high radiation loading wasnecessary throughout in order to acquire images with sufficientbrightness contrast or phase contrast, especially in high-resolutionstructure analyses of extremely low-contrast objects, for example,individual cells, organelles, organisms or parasites.

In order to reduce radiation loading while nevertheless increasing thequality of image evaluation, it is provided, according to the invention,that during the scanning of a line and/or of the object plane 34 thecoupling in of individual spectral components or a plurality of spectralcomponents or of the entire spectrum, as the case may be, isoccasionally interrupted or, alternatively, individual spectralcomponents or a plurality of spectral components are occasionallycoupled in additionally.

The beam-deflecting device 9 remains active continuously during thechange in the spectral composition or intensity of the laser light. Inthis way, for example, locations 36 and 37 within a scanning line orwithin the specimen to be scanned are acted upon differently. Therefore,it is possible for locations 37 which lie within the detail 35 to beevaluated, for example, in a cell, to be subjected to less radiation.

Conversely, an increase in the intensity and/or a change in the spectrumof the laser radiation is carried out during the scanning of location 37when this is desirable, for example, when applying the process accordingto the invention for the purpose of photobleaching, wherein selectedareas of the specimen are to be illuminated with a very high radiationintensity so as to be able to track the dynamic processes taking placeimmediately thereafter.

By means of the process according to the invention and the arrangementaccording to the invention, it is further possible to receive the lightreflected and/or emitted by each individual irradiated location 36 and37 in the individual detection channels 22, wherein each individualdetection channel 22 is modified for receiving different spectralcomponents of the light proceeding from the respective location.

A distinctive feature of the process according to the invention consistsin that the detection and the evaluation of the light proceeding fromevery irradiated location is carried out synchronously with theirradiation of the location in question. To this extent, the excitationwavelength and the emission wavelength can be evaluated for eachindividual location 36 and 37 of the specimen and conclusions can bederived therefrom concerning the characteristics of the specimen atprecisely the observed location.

It is also possible with the arrangement according to the invention tocontinuously monitor the composition and intensity of the laser lightdirected on the specimen based on the signals emitted by theoptoelectronic receiver 31 and to utilize these signals for compensatingfor even very small variations in intensity via the driving device 29.

The excitation radiation and emission radiation which apply to one andthe same location are evaluated by a computing circuit integrated in theevaluation circuit 28. In this way, it can be exactly determined whethera change in the emission wavelength or in the intensity of the emittedradiation which goes beyond a predetermined threshold has taken placeduring the deflection of the laser beam from one location to the other,for example, from directly adjacent locations 36 and 37. If such achange is noted, it may be concluded that an optical boundary layer ispresent in the adjacent locations 36 and 37.

Since the data of the deflection positions in the driving device 29and/or in the evaluation circuit 28 are available for these locations36, 37 and for every other scanned location on the specimen, theconfiguration of optical boundary layers of the type mentioned above canbe determined by the process according to the invention on the basis ofrelevant deflection positions and, finally, the area or volume which isenclosed by the optical boundary layers can be calculated based on thesedeflection positions.

For the sake of completeness, it is noted that the object plane 34 shownin FIG. 2 refers only to one scanning plane of the specimen. It ispossible, of course, to scan a plurality of planes of the specimen inthat the laser radiation is focussed on different coordinates in thez-direction, i.e., vertical to the displayed surface.

While the foregoing description and drawings represent the preferredembodiments of the present invention, it will be obvious to thoseskilled in the art that various changes and modifications may be madetherein without departing from the true spirit and scope of the presentinvention.

REFERENCE NUMBERS

-   1 laser module-   2-4 lasers-   5 beam combiner-   6 AOTF-   7 light-conducting fiber-   8 scanning device-   9 beam-deflecting device-   10 laser module-   11 AOTF-   12 fibers-   13 collimating optics-   14 scanning objective-   15 microscope-   16 specimen-   17 tubelens-   18, 20 beam splitter-   19 microscope objective-   21 imaging optics-   22 detection channels-   23 photomultiplier (pmt)-   24 deflecting prism-   25 dichroitic beam splitter-   26 pinholes-   27 emission filter-   28 evaluating unit-   29 driving device-   30 beam splitter-   31 optoelectronic receiver-   32 line filter-   33 neutral filter-   34 object field-   35 detail-   36, 37 locations-   38 optical axis of the deflected microscope beam path

1. A process for confocal microscopy comprising the steps of: couplingthe laser light of different spectral ranges into a microscope beam pathdeflected in at least two coordinates and directing it successively withrespect to time onto locations of a specimen; permitting the specimen tobe acted upon, location by location and line by line, by the laser lightin at least one plane and generating an image of the scanned plane fromlight returned from the irradiated locations; changing at least one ofthe spectral composition and the intensity of the laser light coupledinto the microscope beam path while the deflection is continued withoutinterruption, so that at least two adjacent locations of the specimenare acted upon by light of at least one of different spectralcharacteristics and different intensity.
 2. The process for confocalmicroscopy according to claim 1, wherein at least one of the spectralcomposition and the intensity of the laser light is changed during thedeflection by occasional additional coupling in of one of individualspectral components and of a plurality of spectral components and byoccasional interruption of the coupling in of individual spectralcomponents and of a plurality of spectral components.
 3. The process forconfocal microscopy according to claim 1, wherein the coupling in of thelaser light is occasionally interrupted during the deflection.
 4. Theprocess for confocal microscopy according to claim 1, wherein at leastone of the spectral composition and intensity of the laser light ischanged during deflection on locations which are located adjacent to oneanother in a line, so that at least locations of this line are actedupon by laser radiation with at least one different spectralcharacteristics and different intensity.
 5. The process for confocalmicroscopy according to claim 2, wherein the locations located adjacentto one another in a line are acted upon repeatedly by the coupled inlaser light and, in this way, always the same locations are exposed tolaser light with different spectral composition and/or with differentintensity.
 6. The process for confocal microscopy according to claim 1,wherein spectral components with wavelengths λ_(A1)=633 nm, λ_(A2)=568nm, λ_(A3)=543 nm, λA_(A4)=514 nm, λ_(A5)=488 nm and/or λ_(A6)=458 nm inthe VIS range and with wavelengths λ_(A7)=351 nm and/or λ_(A8)=364 nm inthe UV range are occasionally coupled in additionally or their couplingin is occasionally interrupted.
 7. The process for confocal microscopyaccording to claim 1, wherein the light returned by every individualirradiated location of the specimen is evaluated with respect to itsspectral characteristics and its intensity, wherein the evaluation iscarried out synchronously in time with the irradiation of the samelocation and while taking into account at least one of the spectralcomposition and intensity of the laser light by which this location wasirradiated.
 8. The process for confocal microscopy according to claim 7,wherein the laser light returned by every individual irradiated locationis detected with a plurality of detection channels, wherein theindividual detection channels are arranged for receiving differentspectral components.
 9. The process for confocal microscopy according toclaim 1, wherein at least one of the spectral composition and theintensity of the laser light which is coupled into the microscope beampath corresponds to the excitation radiation of a fluorescence dyecontained in the specimen or applied to the specimen and the individualdetection channels are configured for the reception of the emissionradiation proceeding from the fluorescence dye.
 10. The process forconfocal microscopy according to claim 1, wherein a mathematical linkingof data characterizing at least one of the spectral composition and theintensity of the laser light directed on a location, of data of theevaluation findings for the light returned by the same location and ofthe deflection positions corresponding to this location is carried outfor the purpose of determining adjustment signals for changing at leastone of the spectral composition and the intensity of the laser lightdirected on this location.
 11. A laser scanning microscope for carryingout a process for confocal microscopy comprising the steps of: couplingthe laser light of different spectral ranges into a microscope beam pathdeflected in at least two coordinates and directing it successively withrespect to time onto locations of a specimen; permitting the specimen tobe acted upon, location by location and line by line, by the laser lightin at least one plane and generating an image of the scanned plane fromlight returned from the irradiated locations; changing at least one ofthe spectral composition and the intensity of the laser light coupledinto the microscope beam path while the deflection is continued withoutinterruption, so that at least two adjacent locations of the specimenare acted upon by light of at least one of different spectralcharacteristics and different intensity, said microscope comprising: alaser module for generating laser light with different selectablespectral components; single-mode fibers for coupling the laser lightinto the microscope beam path; a scanning device which deflects in atleast two dimensions; a microscope objective which focuses the laserlight on a specimen; a plurality of detectors for the reception ofdifferent spectral components of the light returned by the specimen; anevaluation circuit which is connected subsequent to outputs of thedetectors; a plurality of individually controllable single-wavelengthand multiple-wavelength lasers; at least one of a filter which can beinfluenced acousto-optically and an acousto-optic modulator beingprovided in the laser module; photomultipliers being provided asdetectors; color splitters which are arranged on drivable exchangingdevices and which can be substituted for one another being provided forbranching the reflection radiation and emission radiation proceedingfrom the specimen into individual detection channels; and control inputsof the laser module, scanning device and exchanging devices beingconnected with the outputs of the evaluation circuit.
 12. The laserscanning microscope according to claim 11, wherein a beam component ofthe laser light coupled into the microscope beam path is directed on anoptoelectronic receiver whose output is connected with the driving unit.13. The laser scanning microscope according to claim 11, wherein amathematical linking of the output signals of the optoelectronicreceiver with at least one of the output signals of the PMT and thedeflection signals for the scanning device being provided in theevaluation circuit.
 14. A process for confocal microscopy comprising thesteps of: coupling laser light of different spectral ranges into amicroscope beam path deflected in at least two coordinates and directingthe laser light successively with respect to time onto locations of aspecimen; irradiating the specimen, location by location and line byline, by the laser light in at least one plane and generating an imageof the scanned plane from light returned from the irradiated locations;changing both the spectral characteristics and the intensity of thelaser light coupled into the microscope beam path while the deflectioncontinues without interruption, so that at least two adjacent locationsof the specimen are acted upon by light of both different spectralcharacteristics and different intensities.
 15. The process for confocalmicroscopy according to claim 14, wherein the spectral characteristicsand the intensity of the laser light are changed during the deflectionby at least one of (a) occasional additional coupling in of at least oneof ( 1 ) individual spectral components and ( 2 ) a plurality ofspectral components and (b) occasional interruption of the coupling inof at least one of ( 1 ) individual spectral components and ( 2 ) aplurality of spectral components.
 16. The process for confocalmicroscopy according to claim 14, wherein the coupling in of the laserlight is occasionally interrupted during the deflection.
 17. The processfor confocal microscopy according to claim 14, wherein the spectralcharacteristics and intensity of the laser light are changed duringdeflection on locations which are located adjacent to one another in aline, so that at least locations of this line are acted upon by laserradiation with different spectral characteristics and differentintensities.
 18. The process for confocal microscopy according to claim15, wherein the locations located adjacent to one another in a line areacted upon repeatedly by the coupled in laser light and, in this way,the same locations are always exposed to laser light with differentspectral characteristics and different intensities.
 19. The process forconfocal microscopy according to claim 14, wherein spectral componentswith wavelengths λ_(A1) =633 nm, λ _(A2) =568 nm, λ _(A3) =543 nm, λA_(A4) =514 nm, λ _(A5) =488 nm and/or λ _(A6) =458 nm in the VIS rangeand with wavelengths λ _(A7) =351 nm and/or λ _(A8) =364 nm in the UVrange are additionally occasionally coupled in.
 20. The process forconfocal microscopy according to claim 14, wherein the coupling in ofspectral components with wavelengths λ_(A1) =633 nm, λ _(A2) =568 nm, λ_(A3) =543 nm, λA _(A4) =514 nm, λ _(A5) =488 nm and/or λ_(A6) =458 nmin the VIS range and with wavelengths λ _(A7) =351 nm and/or λ _(A8)=364 nm in the UV range is occasionally interrupted.
 21. The process forconfocal microscopy according to claim 14, wherein the light returned byevery individual irradiated location of the specimen is evaluated withrespect to its spectral characteristics and its intensity, and whereinthe evaluation is carried out synchronously in time with the irradiationof the same location and while taking into account at least one of thespectral characteristics and intensity of the laser light by which thislocation was irradiated.
 22. The process for confocal microscopyaccording to claim 21, wherein the laser light returned by everyindividual irradiated location is detected with a plurality of detectionchannels, and wherein the individual detection channels are arranged forreceiving different spectral components.
 23. The process for confocalmicroscopy according to claim 14, wherein at least one of the spectralcharacteristics and the intensity of the laser light which is coupledinto the microscope beam path corresponds to the excitation radiation ofa fluorescence dye contained in the specimen or applied to the specimenand the individual detection channels are configured for the receptionof the emission radiation proceeding from the fluorescence dye.
 24. Theprocess for confocal microscopy according to claim 14, wherein amathematical linking of data characterizing at least one of the spectralcharacteristics and the intensity of the laser light directed on alocation, of data of the evaluation findings for the light returned bythe same location and of the deflection positions corresponding to thislocation is carried out for the purpose of determining adjustmentsignals for changing at least one of the spectral characteristics andthe intensity of the laser light directed on this location.
 25. A laserscanning microscope comprising: means for generating laser light withdifferent spectral components; fibers coupling the laser light into amicroscope beam path; means for deflecting the laser light in at leasttwo dimensions; means for focusing the laser light on a specimen; aplurality of detector means for receiving at least one of (a) differentspectral components of the light returned by the specimen and (b)different spectral components of the light emitted by the specimen, eachdetector means being associated with a detection channel for one of areflection band and an emission band; means for quickly changing atleast one of the spectral characteristics and the intensity of the laserlight during the deflection of the laser light from location to locationin an uninterrupted manner, whereby at least two locations located nextto one another on the specimen are acted upon by at least one of lightwith different spectral characteristics and laser light of differentintensity; means for branching the reflection radiation and emissionradiation proceeding from the specimen into individual detectionchannels; and means for synchronously driving the means for quicklychanging at least one of the spectral characteristics and the intensityof the laser light and the means for deflecting the laser light.
 26. Thelaser scanning microscope according to claim 25, wherein the means forquickly changing includes at least one of (a) means for periodicallyinterrupting coupling in of at least one of ( 1 ) individual spectralcomponents and ( 2 ) a plurality of spectral components and ( 3 ) theradiation of the laser light in its entirety and (b) means forperiodically coupling into the microscope beam path at least one ofindividual spectral components and a plurality of spectral components.27. The laser scanning microscope according to claim 25, wherein themeans for quickly changing at least one of the spectral characteristicsand the intensity of the laser light comprises at least one of a filterwhich can be influenced acousto-optically and an acousto-opticmodulator.
 28. The laser scanning microscope according to claim 25,wherein the different spectral components are selectable.
 29. The laserscanning microscope according to claim 25, wherein the means forbranching comprise color splitters.
 30. The laser scanning microscopeaccording to claim 25, further comprising means for evaluating the lightreturned by every individual irradiated location of the specimen withrespect to its spectral characteristics and its intensity synchronouslyin time with the irradiation of the same location and while taking intoaccount at least one of the spectral characteristics and intensity ofthe laser light by which this location was irradiated.
 31. The laserscanning microscope according to claim 30, further comprising means formathematically linking data characterizing at least one of the spectralcharacteristics and the intensity of the laser light directed on alocation with data from the evaluation means and with the deflectionpositions corresponding to this location, for determining adjustmentsignals for changing at least one of the spectral characteristics andthe intensity of the laser light directed on this location.
 32. A laserscanning microscope comprising: means for generating laser light withdifferent spectral components; fibers coupling the laser light into amicroscope beam path; means for deflecting the laser light in at leasttwo dimensions; means for focusing the laser light on a location of aspecimen; a plurality of detector means for receiving (a) differentspectral components of the light returned by the specimen and (b)different spectral components of the light emitted by the specimen, eachdetector means being associated with a detection channel for one of areflection band and an emission band; means for quickly changing boththe spectral characteristics and the intensity of the laser light whilethe laser light is deflected from location to location in anuninterrupted manner, so that at least two locations located next to oneanother on the specimen are acted upon by light with different spectralcharacteristics and laser light of different intensity; means forbranching the reflection radiation and emission radiation proceedingfrom the specimen into individual detection channels; and means forsynchronously driving the means for quickly changing both the spectralcharacteristics and the intensity of the laser light and the means fordeflecting the laser light.
 33. The laser scanning microscope accordingto claim 32, wherein the means for quickly changing includes at leastone of (a) means for periodically interrupting coupling in of at leastone of ( 1 ) individual spectral components and ( 2 ) a plurality ofspectral components and ( 3 ) the radiation of the laser light in itsentirety and (b) means for periodically coupling into the microscopebeam path at least one of individual spectral components and a pluralityof spectral components.
 34. The laser scanning microscope according toclaim 32, wherein the means for quickly changing both the spectralcharacteristics and the intensity of the laser light comprises at leastone of a filter which can be influenced acousto-optically and anacousto-optic modulator.
 35. The laser scanning microscope according toclaim 32, wherein the different spectral components are selectable. 36.The laser scanning microscope according to claim 32, wherein the meansfor branching comprise color splitters.
 37. The laser scanningmicroscope according to claim 32, further comprising means forevaluating the light returned by every individual irradiated location ofthe specimen with respect to its spectral characteristics and itsintensity synchronously in time with the irradiation of the samelocation and while taking into account at least one of the spectralcharacteristics and intensity of the laser light by which this locationwas irradiated.
 38. The laser scanning microscope according to claim 37,further comprising means for mathematically linking data characterizingthe spectral characteristics and the intensity of the laser lightdirected on a location with data from the evaluation means and with thedeflection positions corresponding to this location, for determiningadjustment signals for changing both the spectral characteristics andthe intensity of the laser light directed on this location.
 39. A laserscanning microscope for carrying out a process for confocal microscopycomprising: means for deflecting a microscope beam in at least twocoordinates and directing the beam successively with respect to timeonto locations of a specimen; means for coupling laser light ofdifferent spectral ranges into the microscope beam; means forirradiating the specimen with the laser light, location by location andline by line, in at least one plane; means for generating an image ofthe scanned plane from light returned from the irradiated locations; andmeans for changing at least one of the spectral characteristics and theintensity of the laser light coupled into the microscope beam path whilethe deflection is continued without interruption, so that at least twoadjacent locations of the specimen are acted upon by light of at leastone of different spectral characteristics and different intensity. 40.The laser scanning microscope according to claim 39, wherein the meansfor changing includes at least one of: means for occasionallyadditionally coupling in of one of individual spectral components and ofa plurality of spectral components and means for occasionallyinterrupting the coupling in of individual spectral components and of aplurality of spectral components.
 41. A laser scanning microscope forcarrying out a process for confocal microscopy comprising: means fordeflecting a microscope beam path in at least two coordinates anddirecting it successively with respect to time onto locations of aspecimen; means for coupling laser light of different spectral rangesinto the microscope beam; means for permitting the laser light to actupon the specimen, location by location and line by line, in at leastone plane; means for generating an image of the scanned plane from lightreturned from the irradiated locations; and means for changing both thespectral characteristics and the intensity of the laser light coupledinto the microscope beam path while the deflection is continued withoutinterruption, so that at least two adjacent locations of the specimenare acted upon by light of both different spectral characteristics anddifferent intensity.
 42. The laser scanning microscope according toclaim 41, wherein the means for changing comprises at least one of:means for occasionally additionally coupling one of individual spectralcomponents and of a plurality of spectral components and means foroccasionally interrupting the coupling in of individual spectralcomponents and of a plurality of spectral components.